Structural basis of G protein mediated signaling

描述（由申请人提供）： 作为囊泡运输的关键调节剂，Rab蛋白包括最大的GTdR家族，在人类基因组中具有37个功能不同的成员和另外20个同种型。与其他GTP酶一样，Rab蛋白在非活性（GDP结合）和活性（GTP结合）状态之间循环。激活和失活分别由鸟嘌呤核苷酸交换因子（GEF）和GTP酶激活蛋白（GAP）调节。一旦被激活，Rab GTP酶与参与囊泡出芽、货物分选、运输、拴系、对接和融合的效应蛋白相互作用。Rab与效应子和调节因子相互作用的特异性被假设在膜运输的保真度方面发挥核心作用。然而，已知越来越多的效应物、GEF和GAP与两种或更多种Rab GTP酶相互作用。此外，Rab GTP酶识别的决定因素是详细的，涉及直接和间接的结构机制。该提案的长期目标是了解与Rab GTP酶的蛋白质-蛋白质相互作用网络的结构基础和机制，以及结构多样的调节因子和效应物如何识别Rab GTP酶。利用基因组测序项目提供的信息，并建立在广泛的初步数据基础上，本提案的目的是从全球、全家族的角度解决识别问题。具体而言，我们将：（目的1）确定活性和非活性构象中尽可能多的Rab GTP酶的高分辨率晶体结构;（目的2）确定整个Rab GTP酶家族的选定效应物和调节因子的特异性;和（目的3）确定效应物和调节因子与在内吞和再循环途径中起作用的Rab GTP酶相互作用的结构基础。结合的结果将提供新的，全家族的洞察力的决定因素识别的Rab GTP酶。Rab GTP酶及其辅助因子的缺陷与几种遗传相关疾病有关，并可能导致包括糖尿病和癌症在内的复杂疾病状态的病理学。因此，Rab辅助因子和调节因子是基于机制的药理学阻断的潜在靶点。

DESCRIPTION (provided by applicant): As critical regulators of vesicular trafficking, Rab proteins comprise the largest GTPase family, with thirty-seven functionally distinct members and another twenty isoforms in the human genome. Like other GTPases, Rab proteins cycle between inactive (GDP bound) and active (GTP bound) states. Activation and inactivation are regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively. Once activated, Rab GTPases interact with effector proteins involved in vesicle budding, cargo sorting, transport, tethering, docking and fusion. The specificity of Rab interactions with effectors and regulatory factors is hypothesized to play a central role with respect to the fidelity of membrane trafficking. However, an increasing number of effectors, GEFs and GAPs are known to interact with two or more Rab GTPases. Moreover, the determinants for recognition of Rab GTPases are elaborate, involving both direct and indirect structural mechanisms. The long range objective of this proposal is to understand the structural bases and mechanisms underlying the network of protein-protein interactions with Rab GTPases and how structurally diverse regulatory factors and effectors recognize Rab GTPases. Capitalizing on the information available from genomic sequencing projects and building on extensive preliminary data, the aims of this proposal address the recognition problem from a global, family wide perspective. Specifically, we will: (Aim 1) determine high resolution crystal structures of as many Rab GTPases as possible in the active and inactive conformations; (Aim 2) determine the specificity of selected effectors and regulatory factors for the entire Rab GTPase family; and (Aim 3) determine the structural bases for interaction of effectors and regulatory factors with Rab GTPases that function in the endocytic and recycling pathways. The combined results will provide novel, family wide insight regarding the determinants for recognition of Rab GTPases. Defects in Rab GTPases and their accessory factors have been implicated in several genetically linked disorders and likely contribute to the pathology of complex disease states including diabetes and cancer. Consequently, Rab accessory and regulatory factors are potential targets for mechanism based pharmacological interdiction.